Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Accumulation kinetics of a series of deletion fragments with or without F6A/F7A substitution

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> () Sequence alignment of the N-terminal portion of RFC140 orthologs from nine eukaryotes. Approximately 10 amino-acid residues are indicated. Red-colored residues are highly conserved among all species, and are indicated as the PIP-box (see text). EGFP-fused fragments 1–369 (), 1–397 (), 1–493 (), 1–733 () and full-length (1–1147, ) having normal (closed squares) or the F6A/F7A mutation (open squares) were transiently expressed in HeLa cells and accumulation was measured as the fold increase of fluorescence intensity. Error bars indicate standard error. Error bars not indicated are smaller than symbols. Pictures represent accumulation of FA mutant fragments, and are taken before and 120 min after laser irradiation. Data were taken from five independent experiments. White arrowheads indicate the laser irradiation region

Cellular localization of endogenous RFC140 and PCNA in asynchronous 293T cells

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> S1, S2 and P2 indicate cytoplasmic, nucleoplasm (soluble nuclear) and chromatin/nuclear matrix (insoluble nuclear) fractions, respectively. MEK2 and ORC2 blots are represented as a markers specific to cytoplasmic and chromatin fractions, respectively

Oxidation resistance 1 functions in the maintenance of cellular survival and genome stability in response to oxidative stress-independent DNA damage

BackgroundDNA damage is generated by various intrinsic and extrinsic sources such as reactive oxygen species (ROS) and environmental mutagens, and causes genomic alterations. DNA damage response (DDR) is activated to induce cell cycle arrest and DNA repair. Oxidation resistance 1 (OXR1) is a protein that defends cells against oxidative stress. We previously reported that OXR1 protein functions in the regulation of G2-phase cell cycle arrest in cells irradiated with gamma-rays, suggesting that OXR1 directly responds to DNA damage.PurposeTo clarify the functions of OXR1 against ROS-independent DNA damage, HeLa and OXR1-depleted HeLa cells were treated with heavy-ion beams and the ROS-independent DNA-damaging agent methyl methanesulfonate (MMS).ResultsFirst, OXR1-depleted cells exhibited higher sensitivity to MMS and heavy-ion beams than control cells. Next, OXR1 depletion increased micronucleus formation and shortened the duration of G2-phase arrest after treatment with MMS or heavy-ion beams. These results suggest that OXR1 functions in the maintenance of cell survival and genome stability in response to DNA damage. Furthermore, the OXR1 protein level was increased by MMS and heavy-ion beams in HeLa cells.ConclusionsTogether with our previous study, the present study suggests that OXR1 plays an important role in the response to DNA damage, not only when DNA damage is generated by ROS

() Recruitment of EGFP-tagged proteins related to DNA repair synthesis to DNA damage induced by a 365-nm UVA-laser

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> (A) Nuclei (arrowheads) of HeLa cells expressing EGFP-RFC140, EGFP-PCNA and EGFP-Polδ1, were irradiated with a 365-nm UVA laser as described in ‘Materials and Methods’ section. () Accumulation of EGFP-fusions, RFC140 (square), PCNA (triangle) and Polδ1 (circle) in (A) was measured as the fold increase of fluorescence intensity at an irradiated site. Data were taken from five independent experiments. Error bars represent standard errors. () Intensity at laser irradiation sites of EGFP-fusions and five RFC subunits was plotted as in (B). () Maximum intensity (MI) and the time to reach MI ( MAX) were represented in each GFP-fusion. A half of MI (1/2 MI) was calculated as 0.5 × (MI − 1) + 1. Thus, 1/2 MI indicates the time to reach 50% of MI

Accumulation of EGFP-RFC140 and EGFP-PCNA in response to laser-induced DNA damage in xrcc1-deficient and xrcc1-proficient mouse embryonic cells

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> Nuclei of xrcc1-deficient (KO) or proficient (WT) mouse embryonic cells expressing EGFP-RFC140 or EGFP-PCNA were irradiated with a low dose (SSBs) or a high dose (SSBs + DSBs) of 365-nm UVA-laser light. Time-lapse pictures were taken as indicated in A